It is a widely adopted sample separation method to selectively bind magnetic labels to biological cells and molecules and utilize the labels' magnetic property to separate the cells and molecules from the biological solution with an externally applied magnetic field. The selective bindings commonly used are polynucleic acid bindings or hybridizations (RNA and DNA), many types of ligand to receptor bindings, as well as antibody to antigen bindings.
With the same biological binding process, it has been a primary purpose of many prior arts to detect the magnetic field emanating from the bound magnetic labels with magneto-resistive (MR) sensors. Thus, the detection and counting of the cells and molecules can be accomplished without using extra step of dye-staining, complex optical imaging components and expensive cytometry systems. In the form of a binding assay, such magnetic detection is expected to achieve single molecule and single cell accuracy. Meanwhile, it has much less complexity and lower cost. Because of the MR sensor is entirely solid state, such magnetic based detection/counting device also shows the promise of small-form-factor hand held capability that enables fast, efficient and economically viable biological and medical applications, such as early detection of cancer and in-field virus or bacteria monitoring.
For molecule detection, binding assay to detect target molecules is already a widely used technique in biological, bio-chemical and medical areas. The target molecules in these bindings, for example, proteins, RNA and DNA, can also be a distinctive component or product of viruses, bacteria and cells, which may be the actual objects of interest for the detection. In a binding assay, the binding molecules are attached to a solid substrate as “capture molecules”. When the assay is exposed to a liquid-form sample, where the target molecules attached to a physical label are contained, the binding molecules capture the target molecules with the specific-bindings and immobilize the target molecules on the surface. This capture process is also called “recognition”. In various previous techniques utilizing the labeled binding process, the labels originally attached to the target molecule are also immobilized on the surface after the recognition process. The labels are either bound together with the target molecules on the surface (“sandwich” assay) or by themselves (“competitive” assay). After removal of the non-specific-binding labels, the bound labels can then be made to generate measurable signals to indicate presence and even population of the target molecules.
For cell detection, specific binding processes usually exist between the biologically coated magnetic labels and the cells, where antibody-antigen binding between the labels and the cells are used. The biological cells can be normal blood cells, body cells, cancer cells and other malignant cells. Cell sizes usually vary from micron size to tens of microns. The antigen binding sites on cells usually vary from thousands to hundreds of thousands number. Magnetic labels as small as sub-micron particles can also be used to form full coverage of the cell surface. Therefore, each cell can be regarded as a single detectable entity. Quantization of different cells not only can serve simple purposes, for example like a normal blood test, but also as a measure of existence of various diseases.
Using MR sensor to detect the magnetic field from the magnetic labels is regarded the most promising method to achieve the goal of on-chip and hand-held molecule and cell counting devices. In prior arts, the MR devices are embedded underneath the binding surface and covered by a protection layer. When the magnetic labels are bound to the surface on the top of a MR sensor, they can generate a magnetic field spontaneously, or, for super-paramagnetic labels, in the presence of an applied magnetic field. This magnetic field from the magnetic labels can then change the MR sensor's resistance state. With a sense current passing through the sensor, a measurable voltage signal can be produced.
The magnetic labels used in previous studies [1-10] or patents [11-13] are usually super-paramagnetic labels or nano-particles that have no magnetic moment at room temperature in the absence of an externally applied magnetic field. Such labels are desired for biological applications because they do not aggregate under zero field conditions. The labels or particles used in these prior arts usually range in size from tens of nanometers to several microns. When the labels attach to a surface after the recognition process, there can be multiple labels attached to a single MR device, one label per device, or one label on multiple devices. However, the sensing mechanism is generally the same. When the magnetic labels are attached to the MR sensor top surface, the field generated by the magnetic moment of the label will either act directly on the MR sensor below it or it can cancel out a portion of the external magnetic field that is acting on the sensor.
FIG. 1 schematically illustrates the basic setup and methodology described above. Magnetic label 10 is attached to the MR sensor surface through the binding pairs 15 after the recognition process. The MR sensor is usually a giant-magneto-resistive (GMR) or a tunneling-magneto-resistive (TMR) device, which includes magnetic free layer 12, non-magnetic spacer layer 13 and magnetic reference layer 14. Spacer 13 is a conductive layer in GMR sensors and a tunneling insulator layer in TMR sensors.
Magnetization of reference layer 14, as represented by Mreference, is fixed in the X axis direction through exchange field from other underneath magnetic layers not shown in the figure. Reference layer magnetization does not change direction under normal magnetic fields. The free layer's magnetization is in the Y axis direction under zero applied field achieved by a bias field Hbias applied in Y axis or by the shape anisotropy of thin film. With a DC current flowing across the device, either in the XY plane or perpendicularly along Z axis, the voltage across the device will change with the MR resistance change and produces a measurable voltage signal.
In prior studies and patents, several detection schemes were used. One commonly used scheme is applying a magnetic field in the transverse direction 14-10, 12-[3], i.e. along the X axis in FIG. 1. This applied field magnetizes the magnetic label along the field direction. The label's magnetic moment produces a magnetic field in the MR sensor below and partially cancels the applied magnetic field acting on the MR sensor. Therefore, under the same applied field conditions, the voltage across the sensor when a label is present differs from when there is no label attached. The presence of the label is detected with this voltage amplitude difference.
Another label sensing scheme, known as BARC [1-3,11], is to apply a DC field perpendicular to the film plane, i.e. along the Z axis direction. This DC field magnetizes the label vertically, the in-plane component of the field generated by the label moment in the MR sensor below, rotating the free layer magnetizations accordingly. If the reference layer magnetization is aligned along the Y axis, or a multi-layer MR structure is used, this rotation will produce a resistance change. It is referred to as a “scissoring mode” [11]. In both schemes, a reference MR sensor to which labels will not attach at any time is always needed as a comparison basis for the voltage change.
Known Prior Art Problems:
Random label binding sites. A key problem facing the current and prior art is that which binding sites get labeled is largely random. The detection scheme described by FIG. 1 assumes that when a label becomes bound to the surface it is directly above the MR sensor. One-on-one binding of this sort is, however, not readily achievable in practice. Almost all the prior art approaches assume that the positioning of the labels, once they have settled on the surface, will be random. The MR sensor is usually made to be similar or larger in size than the labels so as to increase the probability of labels landing on them so the number of labels ending up at any given sensor varies. Additionally, labels may quite likely land on the edge of, or even between, the sensors, This uncontrolled binding of the labels produces signal variations due to the different number of labels on each sensor.
Quantization by signal amplitude: Detection, as described in prior art, is mainly focused on producing a correlation between the number of labels on a MR sensor and the voltage output of that sensor. Given this correlation, the population of the labels can then be estimated from the observed voltage levels. However, as discussed above, the random location of the labels produces an intrinsic signal fluctuation even if the number of labels on each sensor is the same. Additionally, the label itself will always have shape, size and composition variations. Thus, the correlation is further blurred and sensing accuracy is more reduced.
Additionally, since most of the large sensors used in prior arts do not have a hard bias structure to pin the free layer magnetization, for example as in BARC, domain structures are very likely to form at the sensor edges, leading to low frequency large amplitude Barkhausen noise. With all the noise and fluctuation sources acting together, the population estimation accuracy can be severely limited and the fluctuations can become large enough to inhibit practical binding assay applications that are based on the detection of the absolute field strength.
Large sensor size: The size of the individual sensors shown in some of the prior arts is usually quite large—about several microns in size. Although such large sensors have a higher probability that magnetic labels will settle on them, their signal is also significantly reduced. When a label sits on a large sensor, only the label's in-plane field causes free layer magnetization rotation (which is localized at the sensor area right beneath the label). Such fields decrease very quickly towards the sensor's edges. Since the signal is generated by fields anywhere in the full sensor area, the signal produced by a magnetic label will decrease as the sensor size increases.Sensor to sensor signal variation: In the conventional 2-D sensor matrix used in the prior arts, the binding site variations can be partially alleviated by a 2-D signal mapping. With a 2D mapping of the signal amplitude, the label can be located from its signal amplitude. However, such a scheme makes the assumption that each cell has nearly identical response to the magnetic label and that such response can be characterized. For the detection of multiple labels attached to the surface in clusters, the sensor size needs to be much smaller than a single label and the sensor to sensor distance needs to be very small in order to achieve enough sampling of the magnetic field from every label. However, the patterned MR sensors used for assays have intrinsic signal variations between themselves due to fabrication uncertainty. Also, due to the same uncertainty, the sensor cannot be too small or the sensor to sensor signal and response variation will be too large. Additionally, the large size of the transistors used to power each sensor individually limits the maximum sensor areal density. Therefore, a limitation on the spatially resolution of the cell matrix exists. More importantly, since the patterned MR sensors in the assays do not include a hard bias structure, the MR sensors may have large sensitivity fluctuations because of unpinned edges and leads larger sensitivity variations limits the detection accuracy.
In summary, label detection and population counting in the presence of uncontrolled binding processes and amplitude detection are regarded as impediments to achieving the goal of single label and single molecule detection. Detection aided by a 2D mapping of the MR signal is limited by the spatial resolution from the minimal sensor size and sensor to sensor distance. The large sensor to sensor signal variation, sensor intrinsic signal fluctuation due to fabrication and large noise from unbiased MR structures are also serious challenges in the prior art.
To overcome these problems, a method that can avoid the effects of random label distribution on the binding surface and that can eliminate signal fluctuations arising from label location differences is needed. Such a method should not rely on measurement of the absolute label field magnitude to minimize the effect of variation of the label physical size and magnetic property. Label detection with spatial resolution not limited by the sensor spacing is desired. An MR sensor with no free edges is needed for lowering noise levels. Signal sampling at higher frequencies than currently being used in prior arts is also preferred to reduce the effect of low frequency 1/f noise from the sensor as well as other electrical components within the detection system.